Craigiebuckler, Aberdeen, AB15 8QH, UK

“ENDEGRADE”Endophytic bacteria for improving

phytoremediation

Fiona Porteous Moore,

Colin Campbell, Edward Moore

& E.U. partners

ENDEGRADE7 project partners

SCOTLAND BELGIUM IRELAND DENMARK

Macaulay IT Carlow

Institute

LUC VITO DEC-NV DTU NERI (INDUSTRIAL

PARTNER)

PHYTOREMEDIATION

• Ideally plants must have deep roots perennial large water use

• Plants breakdown/volatilise compounds store them in leaves/shoots

• 4 methods phytovolatilisation phytoextraction rhizofiltration phytostabilisation

Problems with some current remediation strategies

• Phytoremediation> pollutants kill the plants> volatilisation through leaves> partial degradation leading to toxic products

• Soil bioremediation> indigenous population overcomes introduced

degraders> nutrient applications often needed> pollutants can be taken up by plants faster than soil

microbes can degrade them

Endegrade - the concept

• enhance plant survival• enhance degradation• reduce volatilisation

Project aims

•Isolate & identify bacteria from Willow and Poplar•Screen for natural degradation potential for target compounds

•Equip endophytic bacteria with degradation plasmids

•Assess re-colonisation and phytoremediation efficiency

•Risk assessment for field use

What is known about endophytic bacteria?

• neutral or beneficial effects on the plant - direct plant growth-promoting activity - N2-fixation - disease suppression - enhanced pest control

• exist in all plant species examined• 103 - 105 cfu/gm plant tissue• highest numbers observed in the roots • bacteria remain localised in specific tissues

• We do not know…

• community dynamics & diversity

• colonisation potential

• plant specific relationships?

Pollutants problematic for phytoremediation...

Compound Fate in plant(toxic, build up, or volatile)

Reference

Phenols Toxic Pfleeger et al., 1991

Chloro-phenols Toxic Pfleeger et al., 1991

TNT Toxic, degraded to amino-dinitrotoluene Thompson et al., 1998

Amino-dinitrotoluene Rather persistent, toxic Thompson et al., 1998

MTBE Volatile Trapp et al., 1994

BTEX Volatile Trapp et al., 1994

TCE Volatile, Build-up of trichloroacetate Trapp et al., 1994

PER Volatile Trapp et al., 1994

Code Plant Location Supplier total noSA Willow shoot LUC 57BSA Willow leaves LUC 24WHV Poplar var Hoogvorst root LUC 14POPHV Poplar var Hoogvorst shoot LUC 8BHV Poplar var Hoogvorst leaves LUC 36WHZD Poplar var Hazendans root LUC 37HZD Poplar var Hazendans shoot LUC 17BHZD Poplar var Hazendans leaves LUC 11VM(415-419) Brassica napus root VITO 5VM(420-427) Brassica napus shoot VITO 8VM(428-433) Brassica napus leaves VITO 6

Total 223

Bacterial endophyte isolates analysed to date

Enterobacter

Bacillus (7%)

Acinetobacter

Flavimonas

Mycetocola Sphingomonas (6%)

Xylophilus

Herbaspirillum

Duganella

Burkholderia (6%)

Delftia (6%)

Klebsiella

Pantoea

Pseudomonas(42%)

Xanthomonas (7%)

Moraxella

Arthrobacter (5%)

AgreiaAerococcus

Paenibacillus

Curtobacterium Micrococcus

Relative frequencies of bacterial isolates, classified to the genus level, on the basis of 16S rDNA sequence analysis

P. azotoformans

2%

P. fulva13%

P. beteli2% P. jessenii

7%

P. plecoglossida

19%

P. rhodesiae2%

P. tolaasii13%

P. frederiks13%

P. veronii11%

P. migulae2%

P. mandelii4%

P. fragi2%

P. putida2%

P. graminis4%

P. stutzeri4%

Relative frequencies of isolates classified as Pseudomonasspecies, based upon 16S rDNA sequence analysis

Poplar Hazendansisolates

Bacillus

Acinetobacter

ArthrobacterEnterobacter

Flavimonas

Herbaspirillum

Klebsiella

Bacillus

Arthrobacter

PaenibacillusXanthomonas

Xylophilus

Blastomonas

PseudomonasSphingomonas

Pseudomonas

Pseudomonas

0 2 4 6 8 10 12 14

Root

shoot

No of isolates

leaves

ROOT SHOOT

LEAF

HERBASPIRILLUMBACILLUS

PSEUDOMONASENTEROBACTER

SPHINGOMONAS

ACINETOBACTER

KLEBSIELLA

XANTHOMONAS

ARTHROBACTER

FLAVIMONAS

PAENIBACILLUS

XYLOPHILUS BLASTOMONAS

Distribution of endophytic bacterial isolates in Poplar

Multivariate analysis - presence / absence of isolates in different species of trees.

Effect of tree species

Napthalene degradation in Pea (proof of concept)Transpiration rate of uninoculated peas when

napthalene applied

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70 80

Time (hrs)

We

igh

t lo

ss

(gra

ms

) 0 mg/l

5mg/l

10mg/l

20mg/l

Transpiration rate when inoculated with endophytic construct

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70 80

Time (hrs)

We

igh

t lo

ss

(g

ram

s) 0 mg/l

5mg/l

10mg/l

20mg/l Work by: Germaine et al, IT Carlow

Plant protection

• Iris & VM1450 - 2,4-D protection (Germaine et al, IT Carlow)

• Pea & PCB degraders - 4-chlorobiphenyl protection (Germaine et al, IT Carlow)

• Lupine & VM1330- Toluene protection, reduced volatilisation, enhanced growth (Barac et al, LUC)

• Toluene & TCE degrader constructs from Brassica napus - to test in plantae degradation (Borremans et al, VITO)

Re-inoculation• K.Germain et al, FEMS Microbiol.Ecol, in press.

• 3 endophytic isolates gfp/Kan marked and re-inoculated into Poplar (original host)

• All strains colonised roots, and 2 strains colonised stems and leaves after 10 weeks

VM1449 (Ps. veronii) colony on root xylem of poplar tree 10 weeks after inoculation x1000

Poplar used in phytoremediation field trial in Belgium

Current work

• Endophytic construct in Poplar exposed to 2,4-D - does in degrade/protect?

> 2,4-D degradation genes & colonisation

• Seasonal community dynamics of endophytes - affects inoculation time/method

> intra/inter species variation, seasonal dynamics, compartmentalisation

Acknowledgements

Macaulay Institute - Renate Wendler, Duncan White

NERI - Denmark - Uli Karlson

DTU - Denmark - Stefan Trapp

VITO - Belgium - Daniel van der Lelie, Brigitte Borremans

LUC- Belgium - Jaco Vangronsveld, Licy Oeyen, Tanja Barac

IT Carlow- Ireland - David Dowling, David Ryan, Keiran Germaine, Elaine Keogh

DEC NV- Belgium - Siegried D’Haene, Gunther de Becker